Synthesis and Antibacterial Activity of Urea and Thiourea Derivatives of Anacardic Acid Mixture Isolated from A Natural Product Cashew Nut Shell Liquid ( CNSL )

Synthesis and antibacterial activity of some novel urea and thiourea derivatives (8a 8k, 9a 9f) of anacardic acid prepared from commercially available anacardic acid which is obtained from natural product Cashew Nut Shell Liquid (CNSL). Compounds (8a 8k, 9a 9f) were tested for Gram positive and Gram negative bacterial cultures. Most of the compounds were showed active compared with standard drug ampicilline.


Introduction
Anacardic acid mixture (1a-d) isolated from a natural product Cashew Nut Shell Liquid (CNSL) which is a by-product of cashew nut industry and these are salicylic acid derivatives with a nonisoprenoid alk(en)yl side chain [1].Kubo et al. reported the separation of anacardic acid into monoene (15:1), diene (15:2) and triene (15:3) by preparative HPLC and tested against cancer cells, and found to show moderate cytotoxic activity on BT-20 breast and HeLa epithelioid cervix carcinoma cells [2].The emergence of drug resistant strains in clinical applications [3][4][5] especially to Gram positive bacteria [6,7] has created a problem of global proportions [8,9].This phenomenon has led in creating novel antibacterial agents distinct from existing classes of compounds.Anacardic acid (pentadecyl salicylic acid) is a phenolic constituent present in Cashew Nut Shell Liquid (CNSL); (Anacardium occidentale L.) and exhibits antimicrobial properties [10,11] which have led to the preparation of various analogues [12,13].Anacardic acid and its derivatives exhibits biological activities like antimicrobial activity [10,11] and soybean lipoxygenase-1 inhibitory activity [14,15].G. C. Reddy et al. reported the synthesis of benzamide derivatives of anacardic acid [16], sildenafil analogues [17], dihydropyridine analogues [18] as calcium channel blockers, isonicotinoylhydrazones for antimycobacterial activity [19] starting from anacardic acid.Recently, a few anacardic acid derivatives exhibited various activities like affect the structure of the enzyme [20], anacardic acid is a specific activator of kinase activity of Aurora Kinase A [21], suppresses expression of nuclear factor-kB regulated gene products leading to potentiation of apoptosis [22] inhibitor of the HAT activity of recombinant Plasmodium falciparum GCN5 [23] and as modulators of histone acetyltransferases [24].
In the present work we wish to report to synthesize novel cell permeable urea and thiourea compounds from cheaply available anacardic acid which was a major constituent of Cashew Nut Shell Liquid (CNSL) natural source to evaluate their biological activity by various antibacterial strains.This report describes the synthesis, spectroscopic identification and antibacterial activity of some novel urea and thiourea anacardic acid derivatives against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes bacterial strains.

Results and Discussion
Here we described the synthesis of various biologically active novel urea and thiourea derivatives using anacardic acid mixture as starting material and various reagents in the given below conditions (Scheme 1).
The anacardic acid mixture (1a-d) was isolated from commercially available CNSL by a reported method [25].Accordingly CNSL was treated with calcium hydroxide, during which anacardic acid present in CNSL becomes calcium anacardate, which was isolated and hydrolyzed with dil.hydrochloric acid to generate anacardic acid ene mixture, which was a mixture of monoene, diene and triene located at (8'), (8', 11') and (8', 11', 14') of the C15 alkyl chain respectively.Saturated anacardic acid was obtained by hydrogenation of the ene mixture of anacardic acid [17] and was further converted to dialkylated compound by reacting with dimethylsulfate in acetonitrile.Dialkylated anacardic acid was reduced to alcohol by treatment with lithium aluminium hydride in tetrahydrofuran and then protected with methane sulphonyl chloride in dichloromethane.Resultant mesylated compound was reacted with sodium azide followed by reduction with Pd/C under H 2 pressure to obtained amine coupled with various isocynate or iso thiocynate in chloroform to obtain compounds (8a -8k, 9a -9f, Scheme 1) of urea and thiourea derivatives were purified by column chromatography to yield title compounds.The structure of urea and thiourea derivatives (8a -8k, 9a -9f) was determined by using different spectroscopic techniques 1 H NMR, IR, Mass.The resulting compounds are screened for their antibacterial activity.
Biological Activity: The urea and thiourea derivatives (8a -8k,9a -9f) were screened for their antibacterial activity [26] against some of the pathogenic bacteria viz.E.coli (MTCC443), P. aeruginosa (MTCC424), S. aureus, (MTCC96) and S. pyogenes (MTCC443) using agar well diffusion method according to the literature protocol [26] The anti-bacterial activity of the analogues was compared with standard drug ampicilline and the results of investigation have been presented in Table 1 and observed that some of the compounds are showed high biological activity.Based on the test results it is evident that several of synthesized anacardic acid analogues possess moderate to good activity against the Gram + ve and Gram -ve bacteria.Of all the compounds prepared entities 8h, 8i, 8j, 9e showed good activity against E. coli, 8b, 8c, 8e and 8f of E. coli MTCC443, and of P. aeruginosa MTCC424, display good to excellent activity while the remaining compounds showed moderate activity.The most active antibacterial agent against Escherichia coli found to be compound 8h, 8i, 8j, and 9e having -CN, -CF 3 and -OCH 3 groups and other compounds in the series exhibited moderate to good activity.The compound 9b, 9c, 9d and 9e showed good activity against almost all the strains and 9f also showed good activity against S. aureus MTCC96 and S. pyogenes MTCC442.This indicates chloro, dichloro, cyano and methoxy substituted compounds showed better activity when compared to other substituted groups.Here seems to be, thiourea substituted novel compounds are exhibiting better activity than urea substituted compounds.The activity depends to some extent on the R substituent, however all the compounds showed antibacterial activity.So other functionalities in the molecule will contribute to the activity as well.It may be suggested that the anacardic acid derivative with a suitable R may lead to a good antibacterial agent against all the Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes bacterial strains.

Conclusion
In summary, the present study describes a convenient and efficient protocol for the synthesis of sulfonamide derivatives by using anacardic acid mixture using various reagents and different conditions.We believe that this procedure is convenient, economic and a user-friendly process for the synthesis of these various novel urea and thiourea compounds from anacardic acid mixture.All compounds structures are supported by physico chemical and IR, NMR, Mass spectral data.Urea and thiourea derivatives were screened for their antibacterial activity against few bacterial strains and observed that some of the compounds are showed more biological activity than standards used.

General Reagents and Equipment
All chemicals and solvents were obtained from Aldrich and Spectrochem., India and used without further purification.Column chromatographic separations were carried out on silica gel 60 -120 mesh size.Melting points were determined in open glass capillaries on a Mel-temp apparatus and are uncorrected.The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm -1 .
The 1 H and 13 C NMR spectra of samples were recorded on a Varian EM-360, NMR spectrometer using TMS as an internal standard in CDCl 3 .The mass spectra were recorded on Jeol JMS-D 300 and Finnigan Mat b at 70 eV with an emission current of 100 µA.
Synthesis of 2-methoxy-6-pentadecyl-benzyl amine (7): A solution of compound 6 (1.5 g, 4.021 mmol) in ethanol (30 ml) was taken into a 500 ml Parr-hydrogenation vessel and added a suspension of 10% Pd/C (250 mg, 10%) in 20 ml of ethanol under argon atmosphere and applied H 2 -pressure (60 psi) for 2 hrs.Reaction mixture was filtered through celite bed and concentrated the filtrate under reduced pressure to obtain 2-methoxy-6-pentadecyl-benzyl amine (7)  Synthesis of urea and thiourea compounds: A solution of amine (1.0 eq) in dry CHCl 3 was taken in seal tube was added isocynate or iso thiocynate and stirred at room temperature for 3 hrs to 8 hrs reaction went to completed distilled off solvent crude compound was purified by column chromatography.
Synthesis of 1-ethyl-3-(2-methoxy-6-pentadecylbenzyl) urea (8a): Using 7 and ethyl isocyanate as starting materials, the title compound 8a was obtained as a white solid (Yield = 63.1%);m.p.114˚C -115˚C; IR (KBr): ν max 3335, 3050, 2967, 2921, 2849, 1622, 1576, 1467, 1260, 1095, 785, 732 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ Antibacterial Bioassay [26]: Urea and thiourea derivatives of Anacardic acid (8a -8k, 9a -9f) were dissolved in dimethyl sulphoxide at 250 μg/ml concentration.The composition of nutrient agar medium was Bactotryptone (10 g), yeast extract (5 g), NaCl (10 g), final pH 7. 4.After 18 h the exponentially growing cultures of the six bacteria in nutrient broth at 37˚C were diluted in sterile broth.From each of these diluted cultures, 1mL was added to 100 mL sterilized and cooled nutrient agar media to give a final bacterial count of 1 × 10 6 cell/ml.The plates were set at room temperature and later dried at 37˚C for 20 hrs.Paper discs (6 mm, punched from Whatmann no.41 paper) were ultraviolet sterilized and used for the assays.Discs were soaked in different concentration of the test solution and placed on the inoculated agar media at regular intervals of 6 -7 cm, care was taken to ensure that excess solution was not on the discs.All the samples were taken in triplicates.The plates were incubated at 37˚C in an inverted fashion.Activity was determined by zones showing complete inhibition (mm).Growth inhibition was calculated with reference to positive control.

Scheme 1 :
Scheme 1: Synthesis of various biologically active urea and thiourea derivatives from anacardic acid mixture.

Table 1 . Antibacterial activity of Urea and Thiourea derivatives of anacardic acid. Name of the Bacteria
(Conc.250 µg/ml) & Inhibition Zone in mm Compound